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SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo.

ABSTRACT: Mitochondrial dysregulation has been implicated in oxidative stress-induced melanocyte destruction in vitiligo. However, the molecular mechanism underlying this process is merely investigated. Given the prominent role of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase Sirtuin3 (SIRT3) in sustaining mitochondrial dynamics and homeostasis and that SIRT3 expression and activity can be influenced by oxidative stress-related signaling, we wondered whether SIRT3 could play an important role in vitiligo melanocyte degeneration by regulating mitochondrial dynamics. Methods: We initially testified SIRT3 expression and activity in normal and vitiligo melanocytes via PCR, immunoblotting and immunofluorescence assays. Then, cell apoptosis, mitochondrial function and mitochondrial dynamics after SIRT3 intervention were analyzed by flow cytometry, immunoblotting, confocal laser microscopy, transmission electron microscopy and oxphos activity assays. Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), immunoblotting and immunofluorescence assays were performed to clarify the upstream regulatory mechanism of SIRT3. Finally, the effect of honokiol on protecting melanocytes and the underlying mechanism were investigated via flow cytometry and immunoblotting analysis. Results: We first found that the expression and the activity of SIRT3 were significantly impaired in vitiligo melanocytes both in vitro and in vivo. Then, SIRT3 deficiency led to more melanocyte apoptosis by inducing severe mitochondrial dysfunction and cytochrome c release to cytoplasm, with Optic atrophy 1 (OPA1)-mediated mitochondrial dynamics remodeling involved in. Moreover, potentiated carbonylation and dampened peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1?) activation accounted for SIRT3 dysregulation in vitiligo melanocytes. Finally, we proved that honokiol could prevent melanocyte apoptosis under oxidative stress by activating SIRT3-OPA1 axis. Conclusions: Overall, we demonstrate that SIRT3-dependent mitochondrial dynamics remodeling contributes to oxidative stress-induced melanocyte degeneration in vitiligo, and honokiol is promising in preventing oxidative stress-induced vitiligo melanocyte apoptosis.

SUBMITTER: Yi X

PROVIDER: S-EPMC6485185 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:BackgroundOxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown.PurposeTo explore whether MC's autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation.MethodsPIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined.ResultsWhen treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens.ConclusionVitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.

Project description:Mitochondria are highly dynamic organelles and undergo constant fission and fusion, which are both essential for the maintenance of cell physiological functions. Dysregulation of dynamin-related protein 1 (Drp1)-dependent mitochondrial dynamics is associated with tumorigenesis and the chemotherapeutic response in hepatocellular carcinoma (HCC). The enzyme cyclooxygenase-2 (COX-2) is overexpressed in most cancer types and correlates with a poor prognosis. However, the roles played by the translocation of mitochondrial COX-2 (mito-COX-2) and the interaction between mito-COX-2 and Drp1 in chemotherapeutic responses remain to be elucidated in the context of HCC. Bioinformatics analysis, paired HCC patient specimens, xenograft nude mice, immunofluorescence, transmission electron microscopy, molecular docking, CRISPR/Cas9 gene editing, proximity ligation assay, cytoplasmic and mitochondrial fractions, mitochondrial immunoprecipitation assay, and flow cytometry analysis were performed to evaluate the underlying mechanism of how mito-COX-2 and p-Drp1Ser616 interaction regulates the chemotherapeutic response via mitochondrial dynamics in vitro and in vivo. We found that COX-2 and Drp1 were frequently upregulated and confer a poor prognosis in HCC. We also found that the proportion of mito-COX-2 and p-Drp1Ser616 was increased in HCC cell lines. In vitro, we demonstrated that the enhanced mitochondrial translocation of COX-2 promotes its interaction with p-Drp1Ser616 via PTEN-induced putative kinase 1 (PINK1)-mediated Drp1 phosphorylation activation. This increase was associated with higher colony formation, cell proliferation, and mitochondrial fission. These findings were confirmed by knocking down COX-2 in HCC cells using CRISPR/Cas9 technology. Furthermore, inhibition of Drp1 using pharmacologic inhibitors (Mdivi-1) or RNA interference (siDNM1L) decreased mito-COX-2/p-Drp1Ser616 interaction-mediated mitochondrial fission, and increased apoptosis in HCC cells treated with platinum drugs. Moreover, inhibiting mito-COX-2 acetylation with the natural phytochemical resveratrol resulted in reducing cell proliferation and mitochondrial fission, occurring through upregulation of mitochondrial deacetylase sirtuin 3 (SIRT3), which, in turn, increased the chemosensitivity of HCC to platinum drugs in vitro and in vivo. Our results suggest that targeting interventions to PINK1-mediated mito-COX-2/p-Drp1Ser616-dependent mitochondrial dynamics increases the chemosensitivity of HCC and might help us to understand how to use the SIRT3-modulated mito-COX-2/p-Drp1Ser616 signaling axis to develop an effective clinical intervention in hepatocarcinogenesis.

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SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo.

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